The invention relates to a microscope. In particular the invention relates to a confocal scanning microscope having a segmenting device.
Furthermore, the invention relates to a segmenting device which is used, in particular, in the abovenamed microscope or confocal microscope.
A microscope generally comprises a light source and a focusing optical system with the aid of which the light from the source is focused onto a pinhole stop. In this case, a beam splitter, a scanning device for beam control, a microscope optical system, a detection stop and detectors for detecting detection and/or fluorescent light are provided.
In a confocal microscope, the illuminating light is mostly coupled in via the beam splitter. The focus of the light beam is moved with the aid of the scanning device in a sample plane. For this purpose, it is customary to use two mirrors which are tilted, the deflection axes mostly being perpendicular to one another, such that one mirror deflects in the X-direction and the other deflects in the Y-direction.
It is also possible in a transmitted-light arrangement for the fluorescent light or the transmitted lightxe2x80x94the transmission of the exciting lightxe2x80x94, for example, to be detected on the condenser side, that is to say on the side of a condenser arranged downstream of the object. The detection-light beam then does not pass via the scanning mirrors to the detector. Such an arrangement is denoted as a non-descanning arrangement.
In order to detect the fluorescent light, there would be a need in the transmitted-light arrangement for a condenser-side detection stop in orderxe2x80x94as in the descanning arrangement describedxe2x80x94to achieve a three-dimensional resolution. In the case of two-photon excitation, however, it is possible to dispense with a condenser-side detection stop, since the probability of excitation is a function of the square of the photon density or the intensity, which is naturally much higher at the focus than in the neighboring regions. The fluorescent light to be detected therefore originates with high probability in overwhelming proportion from the focusing region, and this renders superfluous further differentiation of fluorescence photons from the focusing region from fluorescence photons from the neighboring regions with the aid of a stop arrangement.
Particularly against the background of a yield of fluorescence photons which is low in any case for two-photon excitation, a non-descanning arrangement in which less light is generally lost on the detection-light path is of interest. Nevertheless, because of the lack of marking in living preparations, cell contours, for example, also cannot be detected sufficiently well in the case of this type of observation of fluorescent light, and so it would be desirable to be simultaneously able to observe the transmitted light, which would permit clear conclusions.
A microscope for simultaneously detecting fluorescent and transmitted light is already known to the applicant from an earlier patent application. The known microscope comprises downstream of a condenser a light-splitting device in the form of at least one color beam splitter which spatially separates or splits the fluorescent light from the transmitted light.
For the purpose of transmitted-light contrast microscopy, in the case of the known microscope the segmenting device required for contrast is arranged in the form of a segment stop in a Fourier plane of the transmitted light downstream of the light-splitting device or downstream of the color beam splitter. This requires a long detection-light path downstream of the light-splitting device, which path also has to be set up in addition to the standard microscope equipment.
It is therefore the object of the present invention to specify a microscope having a detection-light path which is as short as possible and can be implemented with the aid of structurally simple means.
The above object is achieved by a microscope which comprises: a light source for illuminating an object to be investigated, an optical device for splitting transmitted light passing through the object and fluorescent light generated in the object, a segmenting device acting exclusively on the transmitted light, wherein the segmenting device is arranged between the object and the optical device.
It is a further object of the present invention to specify a confocal microscope having a detection-light path which is as short as possible and can be implemented with the aid of structurally simple means.
The above object is achieved by a confocal microscope which comprises: a laser light source for illuminating an object to be investigated, an optical device for splitting transmitted light passing through the object and fluorescent light generated in the object, a segmenting device acting on the transmitted light, wherein the segmenting device is arranged between the object and the optical device.
It is an additional object of the invention to provide a segmenting device which allows a detection-light path to be as short as possible and simple to implement.
The above object is achieved by a segmenting device which comprises: a transparent substrate and a color-selective coating formed on said transparent substrate.
The advantage of the invention is, that an optimized arrangement of the segmenting device solves the above object in a surprisingly simple way. For this purpose, the segmenting device is no longer arranged downstream of the light-splitting device, but between the object and the light-splitting device. A detection-light path, already prescribed by the arrangement of the light-splitting device, between the object and the light-splitting device is employed in this case, in addition, by virtue of the arrangement of the segmenting device on this detection-light path. The creation of an additional detection-light path is thereby avoided.
Consequently, the microscope according to the invention has a detection-light path which is as short as possible with the aid of structurally simple means.